Protein crystallography prescreen kit

ABSTRACT

A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

The United States Government has rights in this invention pursuant toContract No. W-7405-ENG-48 between the United States Department ofEnergy and the University of California for the operation of LawrenceLivermore National Laboratory.

BACKGROUND

1. Field of Endeavor

The present invention relates to X-ray crystallography and moreparticularly to macromolecular crystallization screening.

2. State of Technology

U.S. Pat. No. 5,597,457 for a system and method for forming syntheticprotein crystals to determine the conformational structure bycrystallography to George D. Craig, issued Jan. 28, 1997, provides thefollowing background information, “The conformational structure ofproteins is a key to understanding their biological functions and toultimately designing new drug therapies. The conformational structuresof proteins are conventionally determined by x-ray diffraction fromtheir crystals. Unfortunately, growing protein crystals of sufficienthigh quality is very difficult in most cases, and such difficulty is themain limiting factor in the scientific determination and identificationof the structures of protein samples. Prior art methods for growingprotein crystals from super-saturated solutions are tedious andtime-consuming, and less than two percent of the over 100,000 differentproteins have been grown as crystals suitable for x-ray diffractionstudies.”

U.S. Pat. No. 6,267,935 for crystallization media to Wim G. J. Hol,issued Jul. 31, 2001, provides the following background information,“The process of growing biological macromolecule crystals remains,however, a highly empirical process. Macromolecular crystallization isdependent on a host of experimental parameters, including; pH,temperature, the concentration of salts in the crystallization drop, theconcentration of the macromolecule to be crystallized, and theconcentration of the precipitating agent (of which there are hundreds).In particular, the choice of solute conditions in which to grow crystalscontinues to be a matter for empirical determination. Consequently, theability to rapidly and easily generate many crystallization trials isimportant in determining the ideal conditions for crystallization. Thus,there is a need for sets of preformulated crystallization solutions thatcan be used to rapidly and easily generate many crystallization trials.”

SUMMARY

Features and advantages of the present invention will become apparentfrom the following description. Applicants are providing thisdescription, which includes drawings and examples of specificembodiments, to give a broad representation of the invention. Variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those skilled in the art from this descriptionand by practice of the invention. The scope of the invention is notintended to be limited to the particular forms disclosed and theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

X-ray crystallography is a technique that exploits the fact that X-raysare diffracted by crystals. Growing protein crystals of sufficient highquality for X-ray crystallography is very difficult. One embodiment ofthe present invention is a kit for prescreening protein concentrationfor crystallization. The kit includes a multiplicity of vials, amultiplicity of reagents, a multiplicity of sample plates, andinstructions for using the vials, the reagents, and the sample platesfor assessing the proper protein concentration to carry out proteincrystallization. One embodiment of the present invention provides amethod of sample preparation for protein crystallization. It comprisessteps for prescreening samples of a protein in a solution to assess theproper protein/solution concentration to carry out proteincrystallization and X-ray crystallography. A multiplicity ofpre-selected reagents are placed on a multiplicity of sample plates. Amultiplicity of samples of the protein in a solution in varyingconcentrations are placed on the multiplicity of sample plates. Themultiplicity of sample plates containing the multiplicity of reagentsand the samples are incubated. The multiplicity of sample platescontaining the multiplicity of reagents and the samples are examined todetermine which of the sample concentrations are too low and which thesample concentrations are too high. The sample concentrations that areoptimal for protein crystallization are selected and used.

The invention is susceptible to modifications and alternative forms.Specific embodiments are shown by way of example. It is to be understoodthat the invention is not limited to the particular forms disclosed. Theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of the specification, illustrate specific embodiments of theinvention and, together with the general description of the inventiongiven above, and the detailed description of the specific embodiments,serve to explain the principles of the invention.

FIG. 1 shows one embodiment of a kit for prescreening proteinconcentration for crystallization.

FIG. 2 shows some of the equipment that can be used with the kit forprescreening protein concentration for crystallization.

FIG. 3 shows another embodiment of a kit for prescreening proteinconcentration for crystallization.

FIG. 4 shows yet another embodiment of kit for prescreening proteinconcentration for crystallization.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, to the following detailed information,and to incorporated materials; a detailed description of the invention,including specific embodiments, is presented. The detailed descriptionserves to explain the principles of the invention. The invention issusceptible to modifications and alternative forms. The invention is notlimited to the particular forms disclosed. The invention covers allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention as defined by the claims.

Referring now to FIG. 1, a kit for prescreening protein concentrationfor crystallization is illustrated. The kit is designated generally bythe reference numeral 100. The kit 100 includes a multiplicity of vials101 through 109, a multiplicity of reagents 101A through 109A, and amultiplicity of sample plates 110A through 110G. Instructions 111 forusing the vials, the reagents, and the sample plates for assessing theproper protein concentration to carry out protein crystallization areincluded. The vials 101 through 109 containing reagents 101A through109A, Terazaki plate 110 containing sample plates 110A through 110G, andinstructions 111 are packed in a cardboard box 112.

In order to obtain a crystal, the protein molecules must assemble into aperiodic lattice. It is generally desirable to starts with a solution ofthe protein with a fairly high concentration (2-50 mg/ml) and addreagents that reduce the solubility close to spontaneous precipitation.By slow further concentration, and under conditions suitable for theformation of a few nucleation sites, small crystals may start to grow.Often very many conditions have to be tried to succeed. This is done byinitial screening, followed by a systematic optimization of conditions.Crystals should to be a few tenth of a mm in each direction to be usefulfor diffraction experiments.

The kit 100 aids the macromolecular crystal grower in selecting proteinconcentrations suitable for crystallization experiments. Likelihood ofsuccess and overall efficiency increase through selection of promisingprotein concentrations. The kit 100 contains nine 1.5 ml Eppendorfcryo-vials 101 through 109. The nine vials are labeled with cryo-babieslabels.

The kit 100 includes nine different reagents 101A through 109A that areprecisely formulated and consist of high quality chemicals of peranalysis grade to ensure reliable performance. The reagents in kit 100are 101A—50% PEG 4000, 102A—30% PEG 4000, 103A—5% PEG 4000, 104A—3 MAmmonium Sulfate, 105A—2 M Ammonium Sulfate, 106A—0.5 M AmmoniumSulfate, 107A—40% Isopropanol, 108A—30% Isopropanol, 109A—5%Isopropanol. All reagents are aqueous solutions containing 0.1 MTris/HCI buffer at pH 8.5. One ml of each reagent is provided in the 1.5ml Eppendorf cryo-vials 101 through 109.

The kit 100 contains a 70-well Terazaki plate 110. The Terazaki plate110 includes 10 individual row of sample plates 110A through 110G. TheTerazaki plate 110 can be obtained from Robbins Scientific, or Hampton(Order No. 1004-00-0 at Robbins Scientific, or Hampton HR3-120).

The kit 100 contains instructions 111 for using the vials, the reagents,and the sample plates for assessing the proper protein concentration tocarry out protein crystallization are included. The instructions 111include the following statements:

Protein Prescreen setup:

-   1. Set up 3 rows of 3 l μl drops of your protein at the starting    concentration in the Terazaki plate.-   2. Add 1 μl each of the nine PRE-Screen reagents to your protein on    the Terazaki plate (reagent 1-3 to the first row, reagent 4-6 to the    second row, reagent 7-9 to the third row), mix by pipetting up and    down one or two times.-   3. Incubate for at least 1 min —you do not need to cover the plate-   4. View your results under the microscope and note your observations    of each experiment.

How to evaluate your results: If every drop remains clear after 1-5 min,your protein concentration is too low, you should concentrate yourprotein. If on the other hand every drop contains heavy precipitate,your protein concentration is clearly too high, and the protein shouldbe diluted with a suitable buffer. After you have adjusted your proteinconcentration, you should verify by repeating the PRE-Screen thatprecipitation is induced at a modest number of conditions (perhaps 3-6).This tells you that you have a reached a concentration range optimal forcrystallization screening.

Referring now to FIG. 2, some of the equipment that can be used with thekit 100 is shown. The additional equipment includes a pipette suitable201 for pipetting 1 μl volumes, tips 202, and a sample rack 203. A lightmicroscope can be used. The protein crystallography prescreen kit 100 isa reagent kit designed to aid the macromolecular crystal grower in theset up of successful crystallization experiments.

The sample is initially provided as a protein stock solution in minimalbuffer (typically 10 mM buffer and 50 mM KCl and no phosphate), at somereasonably achievable starting concentration. An example of use of theprotein prescreen setup includes the following steps: (1) Set up 3 rowsof 3 l μl drops of the protein at the starting concentration in theTerazaki plate 110, (2) Add 1 μl each of the nine reagents 101A through109A to the protein on the Terazaki plate 110 (reagent 1-3 to the firstrow, reagent 4-6 to the second row, reagent 7-9 to the third row), mixby pipetting up and down one or two times, (3) incubate for at least 1min—it is not necessary to cover the plate, and (4) view the resultsunder the microscope and note observations of each experiment. If everydrop remains clear after 1-5 min, the protein concentration is too low.The protein sample should be concentrated. If on the other hand everydrop contains heavy precipitate, the protein concentration is too high,and the protein should be diluted with a suitable buffer. After theprotein concentration has been adjusted, the test should be verified byrepeating the prescreen test that precipitation is induced at a modestnumber of conditions (perhaps 3-6). This indicates that a concentrationrange has been reached that is optimal for crystallization screening.

The kit 100 is used by macromolecular crystallographers to assess theproper protein concentration to carry out protein crystallization. Thehandling of the kit 100 is easy and comprehensive and results occurwithin minutes. Typically 1 L1 of the kit 10 is mixed with 1A1 ofprotein of various concentrations on the Terasaki plate. The dropsshould be controlled after 1-5 min incubation time under a lightmicroscope. If all nine reagents produce clear drops, the proteinconcentration is too low, if all nine reagents produce heavyprecipitant, turning the drop opaque, the protein concentration is toohigh for protein crystallization to occur. With this method theconcentration range that will most likely lead to successful proteincrystal formation can be determined easily and quickly.

Referring now to FIG. 3, another embodiment of a kit for prescreeningprotein concentration for crystallization is illustrated. Thisembodiment of a kit for prescreening protein concentration forcrystallization is designated generally by the reference numeral 300.X-ray crystallography is a technique that exploits the fact that X-raysare diffracted by crystals. Growing protein crystals of sufficient highquality for X-ray crystallography is very difficult. The kit 300 aidsthe macromolecular crystal grower in selecting protein concentrationssuitable for crystallization experiments.

A 96-well deep-well block 301 is shown in FIG. 3. The 96-well deep-wellblock 301 is pre-arrayed for ease of automation. The 96-well deep-wellblock 301 is included in kit 300. The kit 300 is moderately expanded to12 reagent conditions to be tested against the protein of interest, The12 reagent conditions test 4 broad chemistries at each of threeconcentrations to bracket the solubility behavior of the protein ofinterest against these chemistries. There are 8 copies (1 in each row302 of the deep-well block 301) of the kit 300 for use with as many as 8proteins of interest to be tested simultaneously for high throughputscreening. The block 301 includes long row 303 with 12 spaces. Twelvevials similar to the vials 101 through 109 shown in FIG. 1 are includedin kit 300. The twelve vials contain reagents. The kit 300 also includesa plate containing sample plates and instructions packed in a cardboardbox.

The instructions provide a method of sample preparation for proteincrystallization and X-ray crystallography. The instructions set out anumber of steps including prescreening samples of a protein in asolution to assess the proper protein/solution concentration to carryout protein crystallization; placing a multiplicity of pre-selectedreagents on a multiplicity of sample plates; placing a multiplicity ofsamples of the protein in a solution in varying concentrations on themultiplicity of sample plates; incubating the multiplicity of sampleplates containing the multiplicity of reagents and the samples, viewingthe multiplicity of sample plates containing the multiplicity ofreagents and the samples to determine which of the sample concentrationsare too low and which the sample concentrations are too high, andselecting the sample concentrations that are optimal for proteincrystallization.

Referring now to FIG. 4, another embodiment of a kit for prescreeningprotein concentration for crystallization is illustrated. Thisembodiment of a kit for prescreening protein concentration forcrystallization is designated generally by the reference numeral 400.The lit 400 includes a box 301 of vials, a plate 402, and instructions303. The kit 400 is similar to kit 100 shown in FIG. 1; however, kit 400has 6 vials instead of the 9 vials of kit 100. The physical appearanceof kit 100 and kit 400 be nearly identical; however, there are fewerconditions to test with kit 400 and therefore fewer vials.

X-ray crystallography is a technique that exploits the fact that X-raysare diffracted by crystals. Growing protein crystals of sufficient highquality for X-ray crystallography is very difficult. The kit 400 aidsthe macromolecular crystal grower in selecting protein concentrationssuitable for crystallization experiments. The kit 400 includes amultiplicity of vials 404A, 404B, 404C, etc. containing a multiplicityof reagents 405A, 405B, 405C, etc. and the Terazaki plate 404 containingindividual sample plates.

The instructions 403 provide a method of sample preparation for proteincrystallization and X-ray crystallography. The instructions set out anumber of steps including prescreening samples of a protein in asolution to assess the proper protein/solution concentration to carryout protein crystallization; placing a multiplicity of pre-selectedreagents on a multiplicity of sample plates; placing a multiplicity ofsamples of the protein in a solution in varying concentrations on themultiplicity of sample plates; incubating the multiplicity of sampleplates containing the multiplicity of reagents and the samples, viewingthe multiplicity of sample plates containing the multiplicity ofreagents and the samples to determine which of the sample concentrationsare too low and which the sample concentrations are too high, andselecting the sample concentrations that are optimal for proteincrystallization.

The kit 400 comes as a reduced set of reagents from the kit 100 shown inFIG. 1. The kit 400 is derived from observed correlations betweencrystallization success and results from the kit 100 shown in FIG. 1.Applicants have accumulated data of both the prescreen results and thecrystallization success for proteins and derived a subset of conditionsfrom which crystallization results are best predicted. Applicants haveaccumulated results of the prescreen and subsequent crystallizationtrials of ˜70 protein samples. Prescreen results were checked versus thecrystallization success rate (that means the number of crystallizationexperiments that yielded crystals for a given sample divided by thenumber of experiments set up for that sample) and the prescreenconditions and results were picked that correlate most strongly with thesuccess rate. The advantage is that the prescreen will be morepredictive of crystallization success and it will require less material.New prescreen condition can be introduced specifically to test themagainst crystallization success thereby refining the prescreen overtime.

While the invention may be susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and have been described in detail herein.However, it should be understood that the invention is not intended tobe limited to the particular forms disclosed. Rather, the invention isto cover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the followingappended claims.

1. A method of sample preparation for prescreening a proteinconcentration for crystallization, comprising the steps of: prescreeningsamples of a protein in a solution to assess the proper protein/solutionconcentration to carry out protein crystallization; placing amultiplicity of pre-selected reagents on a multiplicity of sampleplates; placing a multiplicity of samples of said protein in a solutionin varying concentrations on said multiplicity of sample plates;incubating said multiplicity of sample plates containing saidmultiplicity of reagents and said samples; viewing said multiplicity ofsample plates containing said multiplicity of reagents and said samplesto determine which of said sample concentrations are too low and whichsaid sample concentrations are too high; and selecting said sampleconcentrations that are optimal for protein crystallization.
 2. Themethod of sample preparation for prescreening protein concentration forcrystallization of claim 1, wherein said multiplicity of reagents areaqueous solutions containing O.1 M Tris/HCl buffer at pH 8.5.
 3. Themethod of sample preparation for prescreening protein concentration forcrystallization of claim 1, wherein said multiplicity of reagentsinclude a reagent of 50% PEG
 4000. 4. The method of sample preparationfor prescreening protein concentration for crystallization of claim 1,wherein said multiplicity of reagents include a reagent of 30% PEG 4000.5. The method of sample preparation for prescreening proteinconcentration for crystallization of claim 1, wherein said multiplicityof reagents include a reagent of 5% PEG
 4000. 6. The method of samplepreparation for prescreening protein concentration for crystallizationof claim 1, wherein said multiplicity of reagents include a reagent of 3M Ammonium Sulfate.
 7. The method of sample preparation for prescreeningprotein concentration for crystallization of claim 1, wherein saidmultiplicity of reagents include a reagent of 2 M Ammonium Sulfate. 8.The method of sample preparation for prescreening protein concentrationfor crystallization of claim 1, wherein said multiplicity of reagentsinclude a reagent of 0.5 M Ammonium Sulfate.
 9. The method of samplepreparation for prescreening protein concentration for crystallizationof claim 1, wherein said multiplicity of reagents include a reagent of40% Isopropanol.
 10. The method of sample preparation for prescreeningprotein concentration for crystallization of claim 1, wherein saidmultiplicity of reagents include a reagent of 30% Isopropanol.
 11. Themethod of sample preparation for prescreening protein concentration forcrystallization of claim 1, wherein said multiplicity of reagentsinclude a reagent of 5% Isopropanol.
 12. The method of samplepreparation for prescreening protein concentration for crystallizationof claim 1, wherein said multiplicity of reagents include a firstreagent of 50% PEG 4000, a second reagent of 30% PEG 4000, a thirdreagent of 5% PEG 4000, a fourth reagent of 3 M Ammonium Sulfate, afifth reagent of 2 M Ammonium Sulfate, a sixth reagent of 0.5 M AmmoniumSulfate, a seventh reagent of 40% Isopropanol, an eight reagent of 30%Isopropanol, and a ninth reagent of 5% Isopropanol.